In-situ rapid detection method and application for stability of nanoemulsion-based dispersion system

Abstract

The present invention belongs to the technical field of food processing, and particularly relates to an in-situ rapid detection method and application for the stability of a nanoemulsion-based dispersion system. The present invention provides an in-situ rapid detection method for the quality of a nanoemulsion-based dispersion system by combining the structural characteristics of a nanoemulsion with a fluorescence probe of a specific polymer structure. The fluorescence π-conjugated polymer structure probe method used in the present invention has the advantages of simple operation, high speed and efficiency, convenience and time saving, etc., and can be applied to the determination of the stability during the storage of an emulsion dispersion system and / or the prediction of the shelf life of related products.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to an in-situ rapid detection method and application for the stability of a nanoemulsion-based dispersion system. Background Art

[0002] Lipid-soluble active substances such as carotenoids, flavonoids, anthocyanins, and biosaponins commonly found in healthy foods have poor stability, water solubility, and bioavailability, are easily affected by environmental factors and gastrointestinal digestion, and face technical problems such as maintaining activity, increasing stability, improving solubility, enhancing dispersibility, and bioavailability when applied. The commonly used solution is to construct a carrier system, such as an emulsion-based dispersion system, to reduce the influence of external factors. An emulsion-based dispersion system is usually composed of two immiscible liquids, where one liquid is dispersed in the form of droplets in the other liquid. For example, oil droplets are embedded in the aqueous phase to form an oil-in-water emulsion. Common emulsion-based foods include beverages, cakes, ice creams, chocolates, etc. However, from a thermodynamic perspective, an emulsion-based dispersion system is an unstable system and is prone to instability phenomena such as sedimentation, flocculation, coalescence, Ostwald ripening, and creaming during transportation, processing, storage, etc.

[0003] Currently, common methods for measuring the stability of food emulsion-based dispersion systems include charge distribution method, transmission light turbidity method, rheological method, high-speed centrifugation analysis method, etc. Among them, the charge distribution method requires dilution of the sample, which will destroy the original structure of the emulsion-based dispersion system. The transmission light turbidity method and the rheological method are both indirect analyses of the sample. There are significant differences between the experimental results of the high-speed centrifugation analysis method and the actual stability of the sample. Although these detection methods can detect and analyze the quality parameters of food emulsion-based dispersion systems, they cannot intuitively and accurately provide technical references for studying aspects such as the quality and instability mechanism of food emulsion-based dispersion systems.

[0004] Molecular rotors are a class of functional substances that can generate fluorescence signals for specific detection, imaging, etc. under the excitation of an external light source with a specific wavelength; in a low-viscosity solution, they dissipate the excited-state energy through the rotatable structure in the molecule and exhibit very weak fluorescence; while in a high-viscosity solution, the rotation is restricted, and the excited-state energy is dissipated by means of radiative transition, thereby releasing a strong fluorescence signal. Currently, visualization means based on molecular rotors are widely used in the detection of fields such as cell microenvironment, protein recognition, and viscosity of gelatinized starch. However, no report has been retrieved on using a fluorescent π-conjugated polymer structure probe for predicting the stability of a nanoemulsion-based dispersion system. Based on this, the present invention has developed an in-situ rapid detection method and application for predicting the quality of a nanoemulsion-based dispersion system and has conducted detailed research. Summary of the Invention

[0005] Technical Problem:

[0006] A method for in-situ rapid detection of the stability of a nanoemulsion-based dispersion system is provided, which combines the characteristics of the emulsion structure change during the instability process of a heterogeneous oil-in-water nanoemulsion-based dispersion system with a fluorescent π-conjugated polymer structure probe.

[0007] Technical solution:

[0008] The first object of the present invention is to provide an in-situ rapid detection method for the stability of a nanoemulsion-based dispersion system, including the following steps:

[0009] (1) Dissolve the fluorescent π-conjugated polymer structure probe in an organic phase system containing or not containing a fat-soluble active substance to obtain organic phase A, and store it in a low-temperature and light-shielded environment.

[0010] (2) Select a natural biopolymer as an interfacial active substance and dissolve it in an aqueous phase system to obtain aqueous phase B; under an external force field, disperse the organic phase A obtained in step (1) in the form of small droplets in the continuous aqueous phase B to form a heterogeneous oil-in-water nanoemulsion-based dispersion system.

[0011] (3) Store the heterogeneous oil-in-water nanoemulsion-based dispersion system, and at the same time use a fluorescence spectrometer to scan the emission spectra of the heterogeneous oil-in-water nanoemulsion-based dispersion system at different storage times to obtain the corresponding relative fluorescence intensities.

[0012] (4) Predict the stability of the emulsion by analyzing the changes in the relative fluorescence intensities of the heterogeneous oil-in-water nanoemulsion-based dispersion system at different storage times: when the relative fluorescence intensity increases, it indicates that the emulsion stability shows a downward trend; the higher the increment of the relative fluorescence intensity with the storage time, the worse the emulsion stability.

[0013] As a preferred embodiment of the present invention, the fluorescent π-conjugated polymer structure probe includes any one of julolidine derivatives, boron-dipyrromethene derivatives, dialkylaniline derivatives, carbazole derivatives, and benzothiazole derivatives.

[0014] As a preferred embodiment of the present invention, the fluorescent π-conjugated polymer structure probe includes any one of 9-(2,2-dicyanovinyl)julolidine, boron dipyrrolidine, p-dimethylaminobenzonitrile, thioflavin T, 6-propionyl-2-(dimethylamino)naphthalene and its derivatives.

[0015] As a preferred embodiment of the present invention, the concentration of the fluorescent π-conjugated polymer structure probe in organic phase A is 1-60 mM. Further preferably, the concentration of the fluorescent π-conjugated polymer structure probe in organic phase A is 1-8 mM.

[0016] As a preferred embodiment of the present invention, the fat-soluble active substance includes at least one of carotenoids, flavonoids, anthocyanins, tocopherols, terpenoids, saponin compounds, sterols, etc.

[0017] As a preferred embodiment of the present invention, the organic phase system includes any one of triglycerides, corn oil, sweet orange oil, rapeseed oil, linseed oil, fish oil, DHA algal oil, peppermint essential oil, thyme essential oil, and limonene.

[0018] As a preferred embodiment of the present invention, the natural biopolymer includes polysaccharide or protein.

[0019] As a preferred embodiment of the present invention, the polysaccharide includes any one of modified starch, phytoglycogen, cellulose, chitin, and arabic gum.

[0020] As a preferred embodiment of the present invention, the protein includes any one of whey protein and sodium caseinate.

[0021] As a preferred embodiment of the present invention, the mass concentration of the natural biopolymer in aqueous phase B is 1-30%. Further preferably, the mass concentration of the natural biopolymer in aqueous phase B is 2-10%.

[0022] As a preferred embodiment of the present invention, the volume mixing ratio of organic phase A to aqueous phase B is 1:(10-80). Further preferably, the volume mixing ratio of organic phase A to aqueous phase B is 1:(10-50).

[0023] As a preferred embodiment of the present invention, in step (2), the external force field includes at least one of high-pressure homogenization, high-pressure microfluidization, ultrasonic emulsification, microchannel emulsification, membrane emulsification, self-emulsification method, and phase inversion emulsification in common operation processes of the food industry.

[0024] As a preferred embodiment of the present invention, in step (3), the detection conditions of the fluorescence spectrometer are: the excitation wavelength range is 250-550 nm, and the sample detection volume is 0.1-1.0 mL.

[0025] As a preferred embodiment of the present invention, in step (3), the storage conditions are: storing at 0-50 °C for 1-90 d.

[0026] The first object of the present invention is to provide the application of the foregoing method in predicting the storage stability of a nanoemulsion-based dispersion system, which is characterized by including the following steps:

[0027] S1. The same as steps (1)-(3); obtaining the relative fluorescence intensity corresponding to the to-be-detected oil-in-water nanoemulsion-based dispersion system at different storage times;

[0028] S2. Refer to steps (1)-(3), the difference is only that the natural biopolymer in step (2) is replaced with a commercially available starch-based emulsifier, where the storage time and the test conditions of the fluorescence spectrometer in steps S1 and S2 are the same; obtain the relative fluorescence intensities of the heterogeneous oil-in-water nanoemulsion-based dispersion systems prepared from the commercially available starch-based emulsifier at different storage times;

[0029] S3. Substitute the measured relative fluorescence intensities into the following formula to calculate the relative stability parameter of the emulsion S = (FS2 - FS1) / (PS2 - PS1)×100%, where FS2 is the relative fluorescence intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured corresponding to the storage time t2, FS1 is the relative fluorescence intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured corresponding to the storage time t1, PS2 is the relative fluorescence intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system prepared from the commercially available starch-based emulsifier corresponding to the storage time t2, and PS1 is the relative fluorescence intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system prepared from the commercially available starch-based emulsifier corresponding to the storage time t1; the smaller the relative stability parameter S of the emulsion, the more excellent the predicted stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured; when S < 110%, it is predicted that the stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured is good.

[0030] As a preferred embodiment of the present invention, the commercially available starch-based emulsifier includes any one of common commercial modified starches such as cyclodextrin, hydroxypropyl starch, carboxymethyl starch, starch acetate, starch succinate, and starch phosphate.

[0031] Beneficial effects:

[0032] (1) The present invention provides a method for in-situ rapid detection of the stability of a nanoemulsion-based dispersion system by combining the characteristics of the emulsion structure change during the destabilization process of a heterogeneous oil-in-water nanoemulsion-based dispersion system with a fluorescent π-conjugated polymer structure probe. By dissolving the fluorescent π-conjugated polymer structure probe in an organic phase system rich in lipophilic active substances, a heterogeneous oil-in-water nanoemulsion with the fluorescent π-conjugated polymer structure probe in the organic phase layer is prepared. The fluorescence shielding effect generated by the encapsulation of the fluorescent π-conjugated polymer structure probe by natural biopolymers is discovered and utilized. That is, when the heterogeneous oil-in-water nanoemulsion is in a steady state, due to the fluorescence shielding effect, the measured relative fluorescence intensity is relatively low; while when the heterogeneous oil-in-water nanoemulsion gradually destabilizes, the encapsulation of the organic phase by natural biopolymers gradually decreases, and the fluorescent π-conjugated polymer structure probe (and lipophilic active substances) in the organic phase will be gradually released, and the fluorescence shielding effect also decreases accordingly, manifested as a gradual increase in the relative fluorescence intensity. Therefore, the stability of the emulsion can be characterized by detecting the change in the relative fluorescence intensity of the emulsion. Based on this, the method of the present invention can visually quantify the stability and quality of the heterogeneous oil-in-water nanoemulsion-based dispersion system, and further help to explore the spatio-temporal variation law of the nano food emulsion-based dispersion system at the microscopic level, realize the comprehensive and accurate prediction of the quality and stability change law during storage of the emulsion-based nutritional food, contribute to better ensuring and controlling the quality of the emulsion-based nutritional food, and provide a reference for the research on the shelf life and destabilization mechanism of nanoemulsions in aspects such as comprehensive and accurate prediction.

[0033] (2) The method of the present invention has the advantages of simple steps, fast and efficient, and low cost. It can not only in-situ predict the stability of the nanoemulsion-based dispersion system, and can be applied to the determination of the stability during storage of the emulsion dispersion system and / or the prediction of the shelf life of related products; moreover, it can explore the spatio-temporal variation law of the food emulsion dispersion system at the microscopic level, and provide a new evaluation method for the creation of a new generation of emulsion-based nutritional foods.

[0034] (3) The present invention uses a fluorescent π-conjugated polymer structure probe to develop an in-situ rapid detection method for the quality of a nanoemulsion-based dispersion system with natural biopolymers as interfacial active substances. The fluorescent π-conjugated polymer structure probe has the advantages of rapid and sensitive response, small detection dosage, and high degree of automation. The in-situ detection method of the present invention is not affected by environmental factors and has high detection efficiency. For nanoemulsion dispersion systems with different storage periods, their quality parameters can be characterized by fluorescence spectroscopy, with good visual detection effects, and can achieve accurate analysis with multiple application prospects. Description of the Drawings

[0035] Figure 1 Relative fluorescence intensity fluorescence spectra corresponding to the emulsion system sample prepared from phytoglycogen stored at 20 °C for 1 d and 60 d in Example 1. Detailed implementation manners

[0036] The content of the present invention will be further clarified below in conjunction with embodiments, but the content protected by the present invention is not limited to the following embodiments only.

[0037] The commercial modified starch in the following embodiments was purchased from National Starch Company.

[0038] The relative stability parameter S of the emulsion = (FS2 - FS1) / (PS2 - PS1)×100%, where FS2 is the relative fluorescence intensity of the emulsion sample to be measured corresponding to the storage time t2, FS1 is the relative fluorescence intensity of the emulsion sample to be measured corresponding to the storage time t1, PS2 is the relative fluorescence intensity of the emulsion prepared by the commercial starch-based emulsifier corresponding to the storage time t2, and PS1 is the relative fluorescence intensity of the emulsion prepared by the commercial starch-based emulsifier corresponding to the storage time t1. The test method for the relative fluorescence intensity of the emulsion prepared by the commercial starch-based emulsifier is carried out with reference to the test method for the relative fluorescence intensity of the emulsion sample to be measured.

[0039] Example 1

[0040] Dissolve 9-(2,2-dicyanovinyl) julolidine in the corn oil system to obtain the organic phase A, and make the working concentration of 9-(2,2-dicyanovinyl) julolidine in the organic phase A be 3 mM, and store it in a low-temperature and light-proof environment; select phytoglycogen as the surfactant, dissolve it in pure water to obtain the aqueous phase B, and make the mass concentration of phytoglycogen in the aqueous phase B be 2%; mix the organic phase A and the aqueous phase B according to the volume mixing ratio of 1:10 of the organic phase A to the aqueous phase B, and prepare a heterogeneous oil-in-water nanoemulsion-based dispersion system under high-pressure homogenization.

[0041] After the heterogeneous oil-in-water nanoemulsion-based dispersion system is stored at 20°C for a certain period of time (1 d and 60 d), use a fluorescence spectrometer to scan the emission spectrum of 0.5 mL of the emulsion sample to be measured in the excitation wavelength range of 450 - 550 nm.

[0042] After measurement, the relative fluorescence intensities of the emulsion system samples prepared from phytoglycogen in Example 1 corresponding to 1 d and 60 d are 910 a.u. and 6820 a.u. respectively (as Figure 1 shown); by using the same method, the relative fluorescence intensities of the emulsion system samples prepared from commercial cyclized starch corresponding to 1 d and 60 d are measured to be 810 a.u. and 6650 a.u. respectively. According to the relative stability parameter formula of the emulsion, the relative stability parameter is calculated to be 101%, predicting that the stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured in Example 1 is better.

[0043] Example 2

[0044] Dissolve boron dipyrrolidine in the peppermint essential oil system to obtain organic phase A, with the working concentration of boron dipyrrolidine in organic phase A being 8 mM, and store it in a low-temperature and light-proof environment; select whey protein as the surfactant, dissolve it in pure water to obtain aqueous phase B, with the mass concentration of glycogen in aqueous phase B being 6%; mix organic phase A and aqueous phase B according to the volume mixing ratio of organic phase A to aqueous phase B of 1:50, and prepare a heterogeneous oil-in-water nanoemulsion-based dispersion system under ultrasonic emulsification.

[0045] After the heterogeneous oil-in-water nanoemulsion-based dispersion system is stored at 10 °C for a certain period of time (1 d and 90 d), use a fluorescence spectrometer to scan the emission spectrum of 0.2 mL of the emulsion sample to be measured in the excitation wavelength range of 300 - 550 nm.

[0046] After measurement, the relative fluorescence intensities of the emulsion system samples prepared from whey protein in Example 2 corresponding to 1 d and 90 d are 1320 a.u. and 8900 a.u. respectively; the relative fluorescence intensities of the emulsion system samples prepared from commercialized acetylated starch corresponding to 1 d and 90 d are measured by the same method to be 1410 a.u. and 8950 a.u. respectively. According to the relative stability parameter formula of the emulsion, the relative stability parameter is calculated to be 100%, predicting that the stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured in Example 2 is good.

[0047] Example 3

[0048] Dissolve p-dimethylaminobenzonitrile in the triglyceride system to obtain organic phase A, with the working concentration of p-dimethylaminobenzonitrile in organic phase A being 1 mM, and store it in a low-temperature and light-proof environment; select chitin as the surfactant, dissolve it in pure water to obtain aqueous phase B, with the mass concentration of glycogen in aqueous phase B being 10%; mix organic phase A and aqueous phase B according to the volume mixing ratio of organic phase A to aqueous phase B of 1:20, and prepare a heterogeneous oil-in-water nanoemulsion-based dispersion system under ultrasonic emulsification.

[0049] After the heterogeneous oil-in-water nanoemulsion-based dispersion system is stored at 30 °C for a certain period of time (1 d and 25 d), use a fluorescence spectrometer to scan the emission spectrum of 0.1 mL of the emulsion sample to be measured in the excitation wavelength range of 300 - 550 nm.

[0050] After measurement, the relative fluorescence intensities of the emulsion system samples prepared from chitin in Example 3 corresponding to 1d and 25d were 720 a.u. and 2950 a.u., respectively; the relative fluorescence intensities of the emulsion system samples prepared from commercial hydroxypropyl starch corresponding to 1d and 25d measured by the same method were 710 a.u. and 2800 a.u., respectively. According to the relative stability parameter formula of the emulsion, the relative stability parameter was calculated to be 107%, predicting that the stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured in Example 3 was good.

Claims

1. An in-situ rapid detection method for the stability of a nanoemulsion-based dispersion system, characterized in that, It includes the following steps: (1) Dissolve the probe in corn oil, peppermint essential oil or triglyceride to obtain organic phase A, and store it in a low-temperature and light-proof environment; (2) Select a natural biopolymer as a surfactant and dissolve it in pure water to obtain aqueous phase B; under an external force field, disperse the organic phase A obtained in step (1) in the form of small droplets in the continuous aqueous phase B to form a heterogeneous oil-in-water nanoemulsion-based dispersion system; (3) Store the heterogeneous oil-in-water nanoemulsion-based dispersion system, and at the same time use a fluorescence spectrometer to scan the emission spectrum of the heterogeneous oil-in-water nanoemulsion-based dispersion system at different storage times to obtain the corresponding fluorescence emission intensity; (4) Predict the stability of the emulsion by analyzing the change in the fluorescence emission intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system at different storage times: when the fluorescence emission intensity increases, it is judged that the emulsion stability shows a downward trend; the higher the increment of the fluorescence emission intensity with the storage time, the worse the emulsion stability is judged; The probe is any one of 9-(2,2-dicyanovinyl) julolidine, boron dipyrromethene, and p-dimethylaminobenzonitrile; The natural biopolymer is phytoglycogen, whey protein or chitin.

2. The method according to claim 1, characterized in that, The concentration of the probe in organic phase A is 1-8 mM.

3. The method according to claim 1, characterized in that, The volume mixing ratio of organic phase A to aqueous phase B is 1:(10-50).

4. The method according to claim 1, wherein In step (3), the detection conditions of the fluorescence spectrometer are: the excitation wavelength range is 300-550 nm, and the sample detection volume is 0.1-0.5 mL.

5. Use of the method according to any one of claims 1 to 4 in predicting the storage stability of a nanoemulsion-based dispersion system, characterized in that, It includes the following steps: S1. It is the same as steps (1)-(3); obtain the fluorescence emission intensity corresponding to the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured at different storage times; S2. Refer to steps (1)-(3), the difference is only that the natural biopolymer in step (2) is replaced by a commercially available starch-based emulsifier, and the storage time and the fluorescence spectrometer test conditions in steps S1 and S2 are the same; obtain the fluorescence emission intensity corresponding to the heterogeneous oil-in-water nanoemulsion-based dispersion system prepared by the commercially available starch-based emulsifier at different storage times; S3. Substitute the measured fluorescence emission intensity into the following formula to calculate the relative stability parameter of the emulsion S=(FS2-FS1) / (PS2-PS1)×100%, where FS2 is the fluorescence emission intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured corresponding to the storage time t2, FS1 is the fluorescence emission intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured corresponding to the storage time t1, PS2 is the fluorescence emission intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system prepared by the commercially available starch-based emulsifier corresponding to the storage time t2, and PS1 is the fluorescence emission intensity of the heterogeneous oil-in-water nanoemulsion-based dispersion system prepared by the commercially available starch-based emulsifier corresponding to the storage time t1; the smaller the relative stability parameter S of the emulsion, the more excellent the stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured is predicted; when S<110%, it is predicted that the stability of the heterogeneous oil-in-water nanoemulsion-based dispersion system to be measured is good.

6. The application according to claim 5, wherein The commercially available starch-based emulsifiers include any one of cyclodextrin, hydroxypropyl starch, carboxymethyl starch, starch acetate, starch succinate, and starch phosphate.

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